Bis-aryl thiazole derivatives

ABSTRACT

Compounds, compositions and methods are provided that are useful in the treatment or prevention of a condition or disorder mediated by an uncoupling protein. In particular, the compounds of the invention modulate the expression and/or activity of UCP3. The subject compositions are particularly useful in the treatment of obesity and type II diabetes, and associated diseases

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a national phase application of InternationalApplication No. PCT/US02/20447, filed Jun 27, 2002, which claims thebenefit of U.S. Provisional Application No. 60/302,193, filed Jun. 29,2001.

FIELD OF THE INVENTION

The present invention relates to compounds that modulate UCP3 expressionand/or activity and are useful in the treatment of conditions anddisorders mediated by UCP3.

BACKGROUND OF THE INVENTION

A mitochondrial protein called uncoupling protein 1 (UCP1) is thought toplay an important role in the body's regulation of energy utilization.Such regulation provides widespread physiological controls, includingbody weight, appetite, glucose metabolism, temperature, immuneresponses, etc. Mechanistically, UCP1 is thought to create a pathwaythat allows dissipation of the proton electrochemical gradient acrossthe inner mitochondrial membrane in brown adipose tissue, withoutcoupling to any other energy consuming process (for review, see Nicholis& Locke (1984) Physiol. Rev. 64:1-64). Unfortunately, the role of UCP1in physiologies, such as body weight regulation in large adult mammals,such as humans, cattle, pigs, etc., is likely to be limited, since thereis little brown adipose tissue in such animals.

UCP2 is a second, related uncoupling protein that is much more widelyexpressed in large adult mammals (see, e.g. Fleury et al. (1997) Nat.Genet. 15:269-272 and Tartaglia et al. WO 96/05861). Consistent with arole in the regulation of energy utilization in general, and in diabetesand obesity in particular, the UCP2 gene is upregulated in response tofat feeding and maps to regions of the human and mouse genomes linked tohyperinsulinaemia and obesity.

More recently, a third structurally related UCP gene, UCP3 has beencharacterized and found to be preferentially expressed in skeletalmuscle and brown adipose tissues; see Vidal-Puig et al. (1997) Biochem.Biophys. Res. Comm. 235:79-82 and Boss et al. (1997) FEBS Lett.408:39-42. UCP3 has been linked to a number of disorders associated withthe control of energy expenditure, including obesity and diabetes.

The identification of compounds that modulate the activity and/orexpression of UCP3 represents an attractive approach to the developmentof therapeutic agents for the treatment of conditions and disordersassociated with energy utilization.

SUMMARY OF THE INVENTION

The present invention provides methods of using bis-aryl thiazolecompounds and compositions to treat conditions and disorders mediated byUCP3. In particular, the present invention provides methods for treatingobesity and diabetes.

The methods of the invention comprise administering to a subject in needthereof a therapeutically effective amount of a compound of formula (1):

wherein X is selected from the group consisting of CO₂R¹ and C(O)NR¹R²,Y is selected from the group consisting of hydrogen, (C₁-C₄)alkyl,fluoro(C₁-C₄)alkyl, aryl, heteroaryl, halogen, NR³R⁴ and CO₂R³, Z¹ isselected from the group consisting of hydrogen, (C₁-C₄)alkyl andhalogen, and Z² is selected from the group consisting of aryl andheteroaryl, or Z¹ and Z² may be combined to form a fused 6-memberedring. R¹, R², R³ and R⁴ are independently selected from the groupconsisting of hydrogen, (C₁-C₄)alkyl, carboxy(C₁-C₄)alkyl and aryl.

The invention also provides methods for treating a condition or disordermediated by UCP3.

The invention further provides methods for treating a condition ordisorder mediated by a nuclear hormone receptor transcription factor.

The present invention also provides pharmaceutical compositionscomprising a pharmaceutically acceptable carrier or excipient incombination with a compound of formula (I):

wherein X is selected from CO₂R¹ and C(O)NR¹R², Y is selected fromhydrogen, (C₁-C₄)alkyl, fluoro(C₁-C₄)alkyl, aryl, heteroaryl, halogen,NR³R⁴ and CO₂R³, Z¹ is selected from hydrogen, (C₁-C₄)alkyl and halogen,and Z² is selected from aryl and heteroaryl, or Z¹ and Z² may becombined to form a fused 6-membered ring. R¹, R², R³ and R⁴ areindependently selected from hydrogen, (C₁-C₄)alkyl, carboxy(C₁-C₄)alkyland aryl.

The present invention also provides compounds of formula (I):

wherein X is selected from CO₂R¹ and C(O)NR¹R², Y is selected fromhydrogen, (C₁-C₄)alkyl, fluoro(C₁-C₄)alkyl, aryl, heteroaryl, halogen,NR³R⁴ and CO₂R³, Z¹ is selected from hydrogen, (C₁-C₄)alkyl and halogen,Z² is selected from aryl and heteroaryl, or Z¹ and Z² may be combined toform a fused 6-membered ring, and R¹, R², R³ and R⁴ are independentlyselected from hydrogen, (C₁-C₄)alkyl, carboxy(C₁-C₄)alkyl and aryl,provided that the compound is not1-[4-(3-(trifluoromethyl)phenyl)thiazol-2-yl]-5-methylpyrazole-4-carboxylicacid,1-[4-(3-(trifluoromethyl)phenyl)thiazol-2-yl]-5-trifluoromethylpyrazole-4-carboxylicacid,1-[4-(3-methoxyphenyl)thiazol-2-yl]-5-trifluoromethylpyrazole-4-carboxylicacid,1-[4-(4-chlorophenyl)thiazol-2-yl]-5-trifluoromethylpyrazole-4-carboxylicacid,1-[4-(4-nitrophenyl)thiazol-2-yl]-5-trifluoromethylpyrazole-4-carboxylicacid,1-[4-(3-(trifluoromethyl)phenyl)-5-bromothiazol-2-yl]-5-trifluoromethylpyrazole-4-carboxylicacid,1-[4-(3-methylphenyl)thiazol-2-yl]-5-trifluoromethylpyrazole-4-carboxylicacid,1-[4-(4-(trifluoromethyl)phenyl)thiazol-2-yl]-5-trifluoromethylpyrazole-4-carboxylicacid,1-[4-(2-(trifluoromethyl)phenyl)thiazol-2-yl]-5-trifluoromethylpyrazole-4-carboxylicacid,1-[4-(3-chlorophenyl)thiazol-2-yl]-5-trifluoromethylpyrazole-4-carboxylicacid, or1-[4-(3-(trifluoromethyl)phenyl)thiazol-2-yl]-5-phenylpyrazole-4-carboxylicacid.

Unless otherwise indicated, the compounds provided in the above formulasare meant to include pharmaceutically acceptable salts and prodrugsthereof.

Other objects, features and advantages of the present invention willbecome apparent to those skilled in the art from the followingdescription and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 provides the structures of exemplary compounds of formula I.

DETAILED DESCRIPTION OF THE INVENTION

Abbreviations and Definitions

The abbreviations used herein are conventional, unless otherwisedefined.

“UCP3”, as used herein, refers to the UCP3 protein, unless otherwisestated.

The terms “treat”, “treating” and “treatment” refer to a method ofalleviating or abrogating a disease and/or its attendant symptoms.

The terms “prevent”, “preventing” and “prevention” refer to a method ofdecreasing the probability or eliminating the possibility that a diseasewill be contracted.

As used herein, the term “UCP3-mediated condition or disorder” and thelike refers to a condition or disorder characterized by inappropriate,e.g., less than or greater than normal, UCP3 functional activity.Inappropriate UCP3 functional activity might arise as the result ofdecreased UCP3 expression (leading to, e.g., obesity or diabetes) orincreased UCP3 expression. A UCP3-mediated condition or disorder may becompletely or partially mediated by inappropriate UCP3 activity.However, a UCP3-mediated condition or disorder is one in whichmodulation of UCP3 results in some effect on the underlying condition ordisease (e.g., a UCP3 agonist results in some improvement in patientwell-being in at least some patients).

As used herein, the term “nuclear hormone receptor-mediated condition ordisorder” and the like refers to a condition or disorder characterizedby inappropriate, e.g., less than or greater than normal, functionalactivity of a particular nuclear hormone receptor. Inappropriate nuclearhormone receptor functional activity might arise as the result ofdecreased nuclear hormone receptor expression or increased nuclearhormone receptor expression. A nuclear hormone receptor-mediatedcondition or disorder may be completely or partially mediated byinappropriate nuclear hormone receptor activity. However, a nuclearhormone receptor-mediated condition or disorder is one in whichmodulation of nuclear hormone receptor results in some effect on theunderlying condition or disease (e.g., a nuclear hormone receptoragonist results in some improvement in patient well-being in at leastsome patients).

The term “therapeutically effective amount” refers to that amount of thecompound being administered sufficient to prevent development of oralleviate to some extent one or more of the symptoms of the diseasebeing treated.

The term “subject” is defined herein to include animals such as mammals,including, but not limited to, primates (e.g., humans), cows, sheep,goats, horses, dogs, cats, rabbits, rats, mice and the like. Inpreferred embodiments, the subject is a human.

As used herein, “obesity” refers to the excessive accumulation of bodyfat. Obesity may have genetic, environmental (e.g., expending lessenergy than is consumed) and regulatory determinants. Hypertension andlipid disorders, such as, hyperlidemia and coronary artery disease, arecommonly associated with obesity.

As used herein, “diabetes” refers to type II diabetes mellitus(non-insulin-dependent diabetes mellitus or NIDDM). NIDDM ischaracterized by insulin resistance and hyperglycemia. Obesity and lipiddisorders are commonly associated with NIDDM.

The term “modulate” refers to the ability of a compound to increase ordecrease the expression and/or activity of UCP3. Modulation, asdescribed herein, includes the inhibition or activation of UCP3 and/orthe downregulation or upregulation of UCP3 expression, either directlyor indirectly. A modulator preferably downregulates UCP3 expressionand/or inhibits UCP3. More preferably, a modulator upregulates ordownregulates UCP3 expression and/or activates or inhibits UCP3. Mostpreferably, a modulator upregulates UCP3 expression and/or activatesUCP3.

The term “alkyl”, by itself or as part of another substituent, means,unless otherwise stated, a straight or branched chain, or cyclichydrocarbon radical, or combination thereof, which may be fullysaturated, mono- or polyunsaturated and can include di- and multi-valentradicals, having the number of carbon atoms designated (i.e., C₁-C₁₀means one to ten carbons). Examples of saturated hydrocarbon radicalsinclude groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl,t-butyl, isobutyl, sec-butyl, cyclohexyl, (cyclohexyl)methyl,cyclopropylmethyl, homologs and isomers of, for example, n-pentyl,n-hexyl, n-heptyl, n-octyl, and the like. An unsaturated alkyl group isone having one or more double bonds or triple bonds. Examples ofunsaturated alkyl groups include vinyl, 2-propenyl, crotyl,2-isopentenyl, 2-(butadienyl), 2,4-pentadienyl, 3-(1,4-pentadienyl),ethynyl, 1- and 3-propynyl, 3-butynyl, and the higher homologs andisomers. The term “alkyl,” unless otherwise noted, is also meant toinclude those derivatives of alkyl defined in more detail below as“heteroalkyl,” “cycloalkyl” and “alkylene.”

The term “alkylene” by itself or as part of another substituent means adivalent radical derived from an alkane, as exemplified by—CH₂CH₂CH₂CH₂—. Typically, an alkyl group will have from 1 to 24 carbonatoms, with those groups having 10 or fewer carbon atoms being preferredin the present invention. A “lower alkyl” or “lower alkylene” is ashorter chain alkyl or alkylene group, generally having eight or fewercarbon atoms.

Preferably, the term “(C₁-C₄)alkyl” refers to a saturated straight orbranched hydrocarbon chain having from 1 to 4 carbon atoms. Examples ofparticular values for “(C₁-C₄)alkyl” include methyl, ethyl, propyl,2-propyl, butyl, etc. Unless otherwise indicated, the name of a specificalkyl group as used herein, refers to the straight chain or unbranchedisomer.

The term “heteroalkyl,” by itself or in combination with another term,means, unless otherwise stated, a stable straight or branched chain, orcyclic hydrocarbon radical, or combinations thereof, consisting of thestated number of carbon atoms and from one to three heteroatoms selectedfrom the group consisting of O, N, Si and S, and wherein the nitrogenand sulfur atoms may optionally be oxidized and the nitrogen heteroatommay optionally be quaternized. The heteroatom(s) O, N and S may beplaced at any interior position of the heteroalkyl group. The heteroatomSi may be placed at any position of the heteroalkyl group, including theposition at which the alkyl group is attached to the remainder of themolecule. Examples include —CH₂—CH₂—O—CH₃, —CH₂—CH₂—NH—CH₃,—CH₂—CH₂—N(CH₃)—CH₃, —CH₂—S—CH₂—CH₃, —CH₂—CH₂——S(O)—CH₃,—CH₂—CH₂—S(O)₂—CH₃, —CH═CH—O—CH₃, —Si(CH3)3, —CH₂—CH═N—OCH₃, and—CH═CH—N(CH₃)—CH₃. Up to two heteroatoms may be consecutive, such as,for example, —CH₂—NH—OCH₃ and —CH₂—O—Si(CH₃)₃. Also included in the term“heteroalkyl” are those radicals described in more detail below as“heteroalkylene” and “heterocycloalkyl.” The term “heteroalkylene” byitself or as part of another substituent means a divalent radicalderived from heteroalkyl, as exemplified by —CH₂—CH₂—S—CH₂CH₂— and—CH₂—S—CH₂—CH₂—NH—CH₂—. For heteroalkyle groups, heteroatoms can alsooccupy either or both of the chain termini. Still further, for alkyleneand heteroalkylene linking groups, no orientation of the linking groupis implied.

The terms “cycloalkyl” and “heterocycloalkyl”, by themselves or incombination with other terms, represent, unless otherwise stated, cyclicversions of “alkyl” and “heteroalkyl”, respectively. Additionally, forheterocycloalkyl, a heteroatom can occupy the position at which theheterocycle is attached to the remainder of the molecule. Examples ofcycloalkyl include cyclopentyl, cyclohexyl, 1-cyclohexenyl,3-cyclohexenyl, cycloheptyl, and the like. Examples of heterocycloalkylinclude 1-(1,2,5,6-tetrahydropyridyl), 1-piperidinyl, 2-piperidinyl,3-piperidinyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl,tetrahydrofuran-3-yl, tetrahydrothien-2-yl, tetrahydrothien-3-yl,1-piperazinyl, 2-piperazinyl, and the like.

The terms “halo” or “halogen,” by themselves or as part of anothersubstituent, mean, unless otherwise stated, a fluorine, chlorine,bromine, or iodine atom. Additionally, terms such as “fluoroalkyl,” aremeant to include monofluoroalkyl and polyfluoroalkyl. For example, theterm “halo(C₁-C₄)alkyl” is meant to include trifluoromethyl,2,2,2-trifluoroethyl, 4-chlorobutyl, 3-bromopropyl, and the like.

The term “aryl,” employed alone or in combination with other terms(e.g., aryloxy, arylthioxy, arylalkyl) means, unless otherwise stated,an aromatic substituent which can be a single ring or multiple rings (upto three rings) which are fused together or linked covalently. The ringsmay each contain from zero to four heteroatoms selected from N, O, andS, wherein the nitrogen and sulfur atoms are optionally oxidized, andthe nitrogen atom(s) are optionally quaternized. The aryl groups thatcontain heteroatoms may be referred to as “heteroaryl” and can beattached to the remainder of the molecule through a heteroatom.Non-limiting examples of aryl groups include phenyl, 1-naphthyl,2-naphthyl, 4-biphenyl, 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 3-pyrazolyl,2-imidazolyl, 4-imidazolyl, pyrazinyl, 2-oxazolyl, 4-oxazolyl,2-phenyl-4-oxazolyl, 5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl,5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl,2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl,4-pyrimidyl, 5-benzothiazolyl, purinyl, 2-benzimidazolyl, 5-indolyl,1-isoquinolyl, 5-isoquinolyl, 2-quinoxalinyl, 5-quinoxalinyl,3-quinolyl, and 6-quinolyl. Substituents for each of the above notedaryl ring systems are selected from the group of acceptable substituentsdescribed below.

Preferably, the term “aryl” refers to a phenyl or naphthyl group whichis unsubstituted or substituted by one, two or three substitutentsselected independently, described below.

Preferably, the term “heteroaryl” refers to a pyrrolyl, pyrazolyl,imidazolyl, pyrazinyl, oxazolyl, isoxazolyl, thiazolyl, furyl, thienyl,pyridyl, pyrimidyl, benzothiazolyl, purinyl, benzimidazolyl, indolyl,isoquinolyl, quinoxalinyl, quinoxalinyl, quinolyl or quinolyl groupwhich is unsubstituted or substituted by one, two or three substitutentsselected independently, described below.

The term “arylalkyl” is meant to include those radicals in which an arylgroup is attached to an alkyl group (e.g., benzyl, phenethyl,pyridylmethyl and the like) or a heteroalkyl group (e.g., phenoxymethyl,2-pyridyloxymethyl, 3-(1-naphthyloxy)propyl, and the like).

Each of the above terms (e.g., “alkyl,” “heteroalkyl” and “aryl”) aremeant to include both substituted and unsubstituted forms of theindicated radical. Preferred substituents for each type of radical areprovided below.

Substituents for the alkyl and heteroalkyl radicals (including thosegroups often referred to as alkylene, alkenyl, heteroalkylene,heteroalkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, andheterocycloalkenyl) can be a variety of groups selected from: —OR′, ═O,═NR′, ═N—OR′, —NR′R″, —SR′, -halogen, —SiR′R″ R′″, —OC(O)R′, —C(O)R′,—CO₂R¹ (e.g., carboxyl, carboxyalkyl), —CONR′R″ (e.g., carbamoyl,carboxamido), —OC(O)NR′R″, —NR″C(O)R′, —NR′—C(O)NR″R′″, —NR″C(O)2R′,—NH—C(NH₂)═NH, —NR′C(NH₂)═NH, —NH—C(NH₂)═NR′, —S(O)R′, —S(O)₂R′,—S(O)₂NR′R″, —CN and —NO₂ in a number ranging from zero to (2N+1), whereN is the total number of carbon atoms in such radical. R′, R″ and R′″each independently refer to hydrogen, unsubstituted(C₁-C₈)alkyl andheteroalkyl, unsubstituted aryl, aryl substituted with 1-3 halogens,unsubstituted alkyl, alkoxy or thioalkoxy groups, or aryl-(C₁-C₄)alkylgroups. When R′ and R″ are attached to the same nitrogen atom, they canbe combined with the nitrogen atom to form a 5-, 6-, or 7-membered ring.For example, —NR′R″ is meant to include 1-pyrrolidinyl and4-morpholinyl. From the above discussion of substituents, one of skillin the art will understand that the term “alkyl” is meant to includegroups such as haloalkyl (e.g., —CF₃ and —CH₂CF₃) and acyl (e.g.,—C(O)CH₃, —C(O)CF₃, —C(O)CH₂OCH₃, and the like). Preferably, the alkylgroups will have from 0 to 3 substituents.

Similarly, substituents for the aryl and heteroaryl groups are variedand are selected from: -halogen, —OR′, —OC(O)R′, —NR′R″, —SR′, —R′, —CN,—NO₂, —CO₂R′ (e.g., carboxyl, carboxyalkyl), —CONR′R″ (e.g., carbamoyl,carboxamido), —C(O)R′, —OC(O)NR′R″, —NR″C(O)R′, —NR″C(O)2R′, —NR′—C(O)NR″R′″, —NH—C(NH2)═NH, —NR′C(NH₂)═NH, —NH—C(NH₂)═NR′, —S(O)R′,—S(O)₂R′, —S(O)₂NR′R″, —N₃, —CH(Ph)₂, fluoro(C₁-C₄)alkoxy, andfluoro(C₁-C₄)alkyl, in a number ranging from zero to the total number ofopen valences on the aromatic ring system; and where R′, R″ and R′″ areindependently selected from hydrogen, (C₁-C₈)alkyl and heteroalkyl,unsubstituted aryl, (unsubstituted aryl)-(C₁-C₄)alkyl, and(unsubstituted aryl)oxy-(C₁-C₄)alkyl.

Two of the substituents on adjacent atoms of the aryl ring mayoptionally be replaced with a substituent of the formula-T-C(O)—(CH₂)_(q)—U—, wherein T and U are independently —NH—, —O—, —CH₂—or a single bond, and q is an integer of from 0 to 2. Alternatively, twoof the substituents on adjacent atoms of the aryl ring may optionally bereplaced with a substituent of the formula -A-(CH₂)_(r)—B—, wherein Aand B are independently —CH₂—, —O—, —NH—, —S—, —S(O)—, —S(O)₂—,—S(O)₂NR′— or a single and r is an integer of from 1 to 3. One of thesingle bonds of the new ring so formed may optionally be replaced with adouble bond. Alternatively, two of the substituents on adjacent atoms ofthe aryl ring may optionally be replaced with a substituent of theformula —(CH₂)_(s)—X—(CH₂)_(t)—, where s and t are independentlyintegers of from 0 to 3, and X is —O—, —NR′—, —S—, —S(O)—, —S(O)₂—, or—S(O)₂NR′—. The substituent R′- in —NR′— and —S(O)₂NR′— is selected fromhydrogen or unsubstituted (C₁-C₆)alkyl.

As used herein, the term “heteroatom” is meant to include oxygen (O),nitrogen (N), sulfur (S) and silicon (Si).

The term “pharmaceutically acceptable salts” is meant to include saltsof the active compounds which are prepared with relatively nontoxicacids or bases, depending on the particular substituents found on thecompounds described herein. When compounds of the present inventioncontain relatively acidic functionalities, base addition salts can beobtained by contacting the neutral form of such compounds with asufficient amount of the desired base, either neat or in a suitableinert solvent. Examples of pharmaceutically acceptable base additionsalts include sodium, potassium, calcium, ammonium, organic amino, ormagnesium salt, or a similar salt. When compounds of the presentinvention contain relatively basic functionalities, acid addition saltscan be obtained by contacting the neutral form of such compounds with asufficient amount of the desired acid, either neat or in a suitableinert solvent. Examples of pharmaceutically acceptable acid additionsalts include those derived from inorganic acids like hydrochloric,hydrobromic, nitric, carbonic, monohydrogencarbonic, phosphoric,monohydrogenphosphoric, dihydrogenphosphoric, sulfuric,monohydrogensulfuric, hydriodic, or phosphorous acids and the like, aswell as the salts derived from relatively nontoxic organic acids likeacetic, propionic, isobutyric, oxalic, maleic, malonic, benzoic,succinic, suberic, fumaric, mandelic, phthalic, benzenesulfonic,p-tolylsulfonic, citric, tartaric, methanesulfonic, and the like. Alsoincluded are salts of amino acids such as arginate and the like, andsalts of organic acids like glucuronic or galactouronric acids and thelike (see, for example, Berge, S. M., et al. (1977) J. Pharm. Sci.66:1-19). Certain specific compounds of the present invention containboth basic and acidic functionalities that allow the compounds to beconverted into either base or acid addition salts.

The neutral forms of the compounds may be regenerated by contacting thesalt with a base or acid and isolating the parent compound in theconventional manner. The parent form of the compound differs from thevarious salt forms in certain physical properties, such as solubility inpolar solvents, but otherwise the salts are equivalent to the parentform of the compound for the purposes of the present invention.

In addition to salt forms, the present invention provides compoundswhich are in a prodrug form. Prodrugs of the compounds described hereinare those compounds that readily undergo chemical changes underphysiological conditions to provide the compounds of the presentinvention. Additionally, prodrugs can be converted to the compounds ofthe present invention by chemical or biochemical methods in an ex vivoenvironment. For example, prodrugs can be slowly converted to thecompounds of the present invention when placed in a transdermal patchreservoir with a suitable enzyme or chemical reagent. Prodrugs are oftenuseful because, in some situations, they may be easier to administerthan the parent drug. They may, for instance, be bioavailable by oraladministration whereas the parent drug is not. The prodrug may also haveimproved solubility in pharmacological compositions over the parentdrug. A wide variety of prodrug derivatives are known in the art, suchas those that rely on hydrolytic cleavage or oxidative activation of theprodrug. An example, without limitation, of a prodrug would be acompound of the present invention which is administered as an ester (the“prodrug”), but then is metabolically hydrolyzed to the carboxylic acid,the active entity. Additional examples include peptidyl derivatives of acompound of the invention.

Certain compounds of the present invention can exist in unsolvated formsas well as solvated forms, including hydrated forms. In general, thesolvated forms are equivalent to unsolvated forms and are intended to beencompassed within the scope of the present invention. Certain compoundsof the present invention may exist in multiple crystalline or amorphousforms. In general, all physical forms are equivalent for the usescontemplated by the present invention and are intended to be within thescope of the present invention.

Certain compounds of the present invention possess asymmetric carbonatoms (optical centers) or double bonds; the racemates, diastereomers,geometric isomers and individual isomers are all intended to beencompassed within the scope of the present invention.

The compounds of the present invention may also contain unnaturalproportions of atomic isotopes at one or more of the atoms thatconstitute such compounds. For example, the compounds may beradiolabeled with radioactive isotopes, such as for example tritium(³H), iodine-125 (¹²⁵I) or carbon-14 (¹⁴C). All isotopic variations ofthe compounds of the present invention, whether radioactive or not, areintended to be encompassed within the scope of the present invention.

Embodiments of the Invention

UCP3 is an attractive target for anti-obesity and/or anti-diabetic drugdevelopment. Human UCP3 has been described (see GenBank Accession No.P55916). Mouse and bovine UCP3 have also been described (see GenBankAccession Nos. P56501 and O77792, respectively). Regulators of UCP3 geneexpression have been described; see, e.g., U.S. Pat. No. 5,849,581,which reference is incorporated by reference herein. Modulation of UCP3expression and/or activity, e.g., upregulation of UCP3 expression and/oractivation of UCP3, is one approach to altering energy balance, e.g.,increasing energy expenditure relative to energy consumption. Thecompounds of the present invention upregulate UCP3 expression and/oractivity, and thus, are useful in, for example, the treatment orprevention of obesity.

Methods of Use

In one aspect, the present invention provides novel methods for usingcompounds of formula (I):

wherein X is selected from CO₂R¹ and C(O)NR¹R², Y is selected fromhydrogen, (C₁-C₄)alkyl, fluoro(C₁-C₄)alkyl, aryl, heteroaryl, halogen,NR³R⁴ and CO₂R³, Z¹ is selected from hydrogen, (C₁-C₄)alkyl and halogen,Z² is selected from aryl and heteroaryl, or Z¹ and Z² may be combined toform a fused 6-membered ring, and R¹, R², R³ and R⁴ are independentlyselected from hydrogen, (C₁-C₄)alkyl, carboxy(C₁-C₄)alkyl and aryl, andcompositions thereof.

One of skill in the art will understand that a number of structuralisomers are represented by formula I. Preferred isomers are those havingthe structural orientation represented by formula (II):

In one group of preferred embodiments, X is CO₂R¹. In particularlypreferred embodiments, X is CO₂R¹ and R¹ is H. Examples of particularvalues for X within this group of preferred embodiments are carboxyl,and carboxyethyl.

In another group of preferred embodiments, X is C(O)NR¹R². Examples ofparticular values for X within this group are CONH₂, C(O)NHCH₃,C(O)NH—(CH₂)₂—CO₂H and C(O)NH—(CH₂)₃—CO₂H.

In another group of preferred embodiments, Y is hydrogen, (C₁-C₄)alkylor fluoro(C₁-C₄)alkyl. In particularly preferred embodiments, Y is(C₁-C₄)alkyl. Examples of particular values for Y are methyl, ethyl,trifluoromethyl and carboxyl.

In another group of preferred embodiments, Z¹ is hydrogen.

In yet another group of preferred embodiments, Z² is aryl. Particularlypreferred are embodiments in which Z² is phenyl or naphthyl. Examples ofparticular values for Z² within this group of preferred embodiments are2-trifluoromethylphenyl, 3-trifluoromethylphenyl,4-trifluoromethylphenyl, 3,5-bis(trifluoromethyl)phenyl, 3-nitrophenyl,3-methoxyphenyl, 3-chlorophenyl, 4-chlorophenyl, 3,4-dichlorophenyl,biphenyl and 2-naphthyl.

In still another group of preferred embodiments, Z² is heteroaryl.Particularly preferred are embodiments in which Z² is pyridyl orpyrimidinyl.

In another group of preferred embodiments, Z¹ and Z² are combined toform a fused 6-membered ring. Particularly preferred are embodiments inZ¹ and Z² are combined to form a fused benzene ring.

Another group of preferred embodiments has the formula (III):

In compounds of formula III, R¹, Y, Z¹ and Z² have the meanings andpreferred groupings provided above.

Yet another group of preferred embodiments is represented by the formula(IV):

In compounds of formula IV, W is (C₁-C₄)alkyl, fluoro(C₁-C₄)alkyl,halogen or nitro, n is an integer from 1-3 and R¹ and Y have themeanings and preferred groupings provided above.

Still another group of preferred embodiments is represented by theformula (V):

wherein R¹, W and n have the meanings and preferred groupings providedabove. An exemplary compound of formula V is:

Still another group of preferred embodiments is represented by theformula (VI):

wherein R¹, R², Y, Z¹ and Z² have the meanings and preferred groupingsprovided above.

Still another group of preferred embodiments is represented by theformula (VII):

In compounds of formula VII, W is (C₁-C₄)alkyl, fluoro(C₁-C₄)alkyl,halogen or nitro, n is an integer from 1-3 and R¹ and Y have themeanings and preferred groupings provided above.

Yet another group of preferred embodiments is represented by the formula(VIII):

wherein R³, R⁴, W and n have the meanings and preferred groupingsprovided above. An exemplary compound of formula VIII is:

In particular, the invention provides novel methods for treating orpreventing prevent a condition or disorder mediated by UCP3, such asobesity or diabetes. The subject methods may also be useful for treatingor preventing a disease, disorder, dysfunction and the like, in whichobesity or diabetes contributes to the pathogenesis thereof, such asatherosclerosis. The subject methods may also be useful for improvinginsulin sensitivity and/or reducing blood glucose, and reducing serumcholesterol levels. The methods typically involve administering to apatient a therapeutically effective amount of one or more of thecompounds of formula I, II, III, IV, V, VI, VII or VIII, or compositionsthereof.

The present invention also provides novel methods for using compounds offormula I, II, III, IV, V, VI, VII or VIII and compositions thereof tomodulate UCP3. The methods typically involve contacting a cell with oneor more of the subject compounds or compositions.

It is believed that the compounds of the invention modulate UCP3expression and/or activity by specifically modulating UCP3 genetranscription. Transcription regulates UCP3 gene expression and isactivated by the association of one or more transcription factors with aUCP3 transcriptional promoter. Therefore, modulating gene transcription,e.g., modulating the association of a transcription factor with a UCP3transcriptional promoter, will modulate UCP3 gene expression and treator prevent a UCP3-mediated condition or disorder. For example, enhancingthe association of a transcription factor with a UCP3 transcriptionalpromoter will increase UCP3 gene transcription and upregulate UCP3 geneexpression, leading to increased UCP3 transcript and/or protein levels(i.e., increased UCP3 mRNA and/or increased UCP3). The resultingincreased UCP3 transcript and/or protein levels can be used to treat asubject deficient in functional UCP3.

While a precise understanding of the mechanism by which the compounds ofthe present invention modulate UCP3 gene transcription is not requiredin order to practice the invention, it is believed that the compoundsinteract, either directly or indirectly, with a transcription factor andmodify the ability of the transcription factor to associate with theUCP3 promoter. In particular, it is believed that the compounds of theinvention interact with a nuclear hormone receptor family transcriptionfactor and modulate its activity.

Accordingly, the present invention provides novel methods for using theforegoing compounds and compositions to treat or prevent conditions anddisorders mediated by a nuclear hormone receptor transcription factor,preferably retinoid X receptor (RXR). Nuclear hormone receptortranscription factors are soluble protein receptors that bind tospecific cis-acting sequences in the promoter region of target genes andmodulate gene expression in response to endogenous hormone activators orligands. Nuclear hormone receptors have been shown to be involved innumerous pathologies, including metabolic diseases, cardiovasculardiseases, lipid disorders and cell proliferative disorders. ExemplaryRXR-mediated conditions and disorders include, but are not limited to,diabetes, atherosclerosis, hyperlipidemia, hypercholesterolemia, cellproliferative disorders such as cancers of the breast, skin, prostate,cervix, uterus, colon, bladder, esophagus, stomach, lung, larynx, oralcavity, blood and lymphatic system, skin proliferative disorders (e.g.,psoriasis) and diseases of the eye (e.g., proliferativevitreoretinopathy).

The present invention further provides novel methods for using theforegoing compounds and compositions to modulate RXR. The methodstypically involve contacting a cell with one or more of the subjectcompounds or compositions.

Compositions

In another aspect, the present invention provides pharmaceuticalcompositions which are suitable for pharmaceutical or diagnostic use.The compositions comprise compounds of formula I, II, III, IV, V, VI,VII or VIII, in combination with a diagnostically or pharmaceuticallyacceptable carrier or excipient. The subject compositions are useful fortreating or preventing conditions and disorders mediated by UCP3, suchas obesity and diabetes. The compounds of the present invention can beprepared and administered in a wide variety of oral and parenteraldosage forms. Thus, the compounds of the present invention can beadministered by injection, that is, intravenously, intramuscularly,intracutaneously, subcutaneously, intraduodenally or intraperitoneally.Also, the compounds described herein can be administered by inhalation,for example, intranasally. Additionally, the compounds of the presentinvention can be administered transdermally. Accordingly, the presentinvention also provides pharmaceutical compositions comprising apharmaceutically acceptable carrier or excipient and either a compoundof formula I, II, III, IV, V, VI, VII or VIII or a pharmaceuticallyacceptable salt of a compound of formula I, II, III, IV, V, VI, VII orVIII.

For preparing pharmaceutical compositions from the compounds of thepresent invention, pharmaceutically acceptable carriers can be eithersolid or liquid. Solid form preparations include powders, tablets,pills, capsules, cachets, suppositories, and dispersible granules. Asolid carrier can be one or more substances which may also act asdiluents, flavoring agents, binders, preservatives, tabletdisintegrating agents, or an encapsulating material.

In powders, the carrier is a finely divided solid which is in a mixturewith the finely divided active component. In tablets, the activecomponent is mixed with the carrier having the necessary bindingproperties in suitable proportions and compacted in the shape and sizedesired.

The powders and tablets preferably contain from 5% or 10% to 70% of theactive compound. Suitable carriers are magnesium carbonate, magnesiumstearate, talc, sugar, lactose, pectin, dextrin, starch, gelatin,tragacanth, methylcellulose, sodium carboxymethylcellulose, a lowmelting wax, cocoa butter, and the like. The term “preparation” isintended to include the formulation of the active compound withencapsulating material as a carrier providing a capsule in which theactive component with or without other carriers, is surrounded by acarrier, which is thus in association with it. Similarly, cachets andlozenges are included. Tablets, powders, capsules, pills, cachets, andlozenges can be used as solid dosage forms suitable for oraladministration.

For preparing suppositories, a low melting wax, such as a mixture offatty acid glycerides or cocoa butter, is first melted and the activecomponent is dispersed homogeneously therein, as by stirring. The moltenhomogeneous mixture is then poured into convenient sized molds, allowedto cool, and thereby to solidify.

Liquid form preparations include solutions, suspensions, and emulsions,for example, water or water/propylene glycol solutions. For parenteralinjection, liquid preparations can be formulated in solution in aqueouspolyethylene glycol solution.

Aqueous solutions suitable for oral use can be prepared by dissolvingthe active component in water and adding suitable colorants, flavors,stabilizers, and thickening agents as desired. Aqueous suspensionssuitable for oral use can be made by dispersing the finely dividedactive component in water with viscous material, such as natural orsynthetic gums, resins, methylcellulose, sodium carboxymethylcellulose,and other well-known suspending agents.

Also included are solid form preparations which are intended to beconverted, shortly before use, to liquid form preparations for oraladministration. Such liquid forms include solutions, suspensions, andemulsions. These preparations may contain, in addition to the activecomponent, colorants, flavors, stabilizers, buffers, artificial andnatural sweeteners, dispersants, thickeners, solubilizing agents, andthe like.

The pharmaceutical preparation is preferably in unit dosage form. Insuch form the preparation is subdivided into unit doses containingappropriate quantities of the active component. The unit dosage form canbe a packaged preparation, the package containing discrete quantities ofpreparation, such as packeted tablets, capsules, and powders in vials orampoules. Also, the unit dosage form can be a capsule, tablet, cachet,or lozenge itself, or it can be the appropriate number of any of thesein packaged form.

The quantity of active component in a unit dose preparation may bevaried or adjusted from 0.1 mg to 1000 mg, preferably 1.0 mg to 100 mgaccording to the particular application and the potency of the activecomponent. The composition can, if desired, also contain othercompatible therapeutic agents.

In therapeutic use for the treatment of obesity or diabetes, thecompounds utilized in the pharmaceutical method of the invention areadministered at the initial dosage of about 0.001 mg/kg to about 100mg/kg daily. A daily dose range of about 0.1 mg/kg to about 10 mg/kg ispreferred. The dosages, however, may be varied depending upon therequirements of the patient, the severity of the condition beingtreated, and the compound being employed. Determination of the properdosage for a particular situation is within the skill of thepractitioner. Generally, treatment is initiated with smaller dosageswhich are less than the optimum dose of the compound. Thereafter, thedosage is increased by small increments until the optimum effect underthe circumstances is reached. For convenience, the total daily dosagemay be divided and administered in portions during the day, if desired.

The compositions may be advantageously combined and/or used incombination with agents useful in the treatment and/or prevention ofobesity and/or diabetes and pathologies associated therewith (e.g.,hyperlipidemia, atherosclerosis). In many instances, administration ofthe subject compounds or compositions in conjunction with thesealternative therapeutic agents enhances the efficacy of such agents.Accordingly, in some instances, the present compounds, when combined oradministered in combination with anti-obesity and/or anti-diabeticagents, can be used in dosages which are less than the expected amountswhen used alone, or less than the calculated amounts for combinationtherapy.

Suitable agents for combination therapy include those that are currentlycommercially available and those that are in development or will bedeveloped. Exemplary agents useful in the treatment of obesity and/ordiabetes include β₃ adrenergic receptor agonists, leptin or derivativesthereof, neuropeptide Y antagonists, insulin and derivatives thereof,hypoglycemic agents, such as sulfonylureas (e.g., meglinatide,tolbutamide, chlorpropamide, acetohexamide, tolazamide, glyburide,glipizide and glimepiride), antihyperglycemic agents, such as biguanides(e.g., metformin), α-glucosidase inhibitors (e.g., acarbose), insulinsensitizers, such as thiazolidinones (e.g., rosiglitazone (Avandia®),troglitazone (Rezulin®) and pioglitazone (Actos®)), and RXR agonists,such as bexarotene (Targretin®).

Exemplary agents useful in the treatment of atherosclerosis andcomplications thereof include cholesterol lowering agents such asHMG-CoA reductase inhibitors (e.g., lovastatin, simvastatin,pravastatin, fluvastatin, atorvastatin and other statins), bile acidsequestrants (e.g., cholestyramine and colestipol), nicotinic acid(niacin), fibric acid derivatives (gemfibrozil, clofibrate, fenofibrateand benzafibrate), probucol and nitroglycerin, and calcium channelblockers (e.g., veraparnil, nicardipine, amlodipine, diltiazem andnifedipine).

Exemplary agents useful in the treatment of cell proliferative disordersand/or skin proliferative disorders include preparations of interferonalpha, e.g., interferon α 2b, and interferon beta, e.g., interferon β-1α (Avonex®) and interferon β-1 β (Betaseron®), DNA-alkylating agents(e.g., cyclophosphamide, ifosfamide), antimetabolites (e.g.,azathioprene, 6-mercaptopurine, methotrexate, a folate antagonist, and5-fluorouracil, a pyrimidine antagonist), microtubule disruptors (e.g.,vincristine, vinblastine, paclitaxel and coichicine), DNA intercalators(e.g., doxorubicin, daunomycin and cisplatin), DNA synthesis inhibitorssuch as hydroxyurea and hormone therapy (e.g., tamoxifen and flutamide)and RXR agonists, such as bexarotene (Targretin®).

Compounds

In another aspect, the present invention provides compounds which arerepresented by the formula (I):

wherein X is selected from CO₂R¹ and C(O)NR¹R², wherein R¹, R², R³ andR⁴ are independently selected from hydrogen, (C₁-C₄)alkyl,carboxy(C₁-C₄)alkyl and aryl, Y is selected from hydrogen, (C₁-C₄)alkyl,fluoro(C₁-C₄)alkyl, aryl, heteroaryl, halogen, NR³R⁴ and CO₂R³, Z¹ isselected from hydrogen, (C₁-C₄)alkyl and halogen, and Z² is selectedfrom aryl and heteroaryl, or Z¹ and Z² may be combined to form a fused6-membered ring, with the proviso that the compound is not

1-[4-(3-(trifluoromethyl)phenyl)thiazol-2-yl]-5-methylpyrazole-4-carboxylicacid (1)

1-[4-(3-(trifluoromethyl)phenyl)thiazol-2-yl]-5-trifluoromethylpyrazole-4-carboxylicacid (5),

1-[4-(3-methoxyphenyl)thiazol-2-yl]-5-trifluoromethylpyrazole-4-carboxylicacid,

1-[4-(4-chlorophenyl)thiazol-2-yl]-5-trifluoromethylpyrazole-4-carboxylicacid,

1-[4-(4-nitrophenyl)thiazol-2-yl]-5-trifluoromethylpyrazole-4-carboxylicacid,

1-[4-(3-(trifluoromethyl)phenyl)-5-bromothiazol-2-yl]-5-trifluoromethylpyrazole-4-carboxylicacid,

1-[4-(3-methylphenyl)thiazol-2-yl]-5-trifluoromethylpyrazole-4-carboxylicacid,

1-[4-(4-(trifluoromethyl)phenyl)thiazol-2-yl]-5-trifluoromethylpyrazole-4-carboxylicacid,

1-[4-(2-(trifluoromethyl)phenyl)thiazol-2-yl]-5-trifluoromethylpyrazole-4-carboxylicacid,

1-[4-(3-chlorophenyl)thiazol-2-yl]-5-trifluoromethylpyrazole-4-carboxylicacid, or

1-[4-(3-(trifluoromethyl)phenyl)thiazol-2-yl]-5-phenylpyrazole-4-carboxylicacid.

One of skill in the art will understand that a number of structuralisomers are represented by formula I. Preferred isomers are those havingthe structural orientation represented by formula (II):

In one group of preferred embodiments, X is CO₂R′. In particularlypreferred embodiments, X is CO₂R¹ and R¹ is H. Examples of particularvalues for X within this group of preferred embodiments are carboxyl,and carboxyethyl.

In another group of preferred embodiments, X is C(O)NR¹R². Examples ofparticular values for X within this group are CONH₂, C(O)NHCH₃,C(O)NH—(CH₂)₂—CO₂H and C(O)NH—(CH₂)₃—CO₂H.

In another group of preferred embodiments, Y is hydrogen, (C₁-C₄)alkylor fluoro(C₁-C₄)alkyl. In particularly preferred embodiments, Y is(C₁-C₄)alkyl. Examples of particular values for Y are methyl, ethyl,trifluoromethyl and carboxyl.

In another group of preferred embodiments, Z¹ is hydrogen.

In yet another group of preferred embodiments, Z² is aryl. Particularlypreferred are embodiments in which Z² is phenyl or naphthyl. Examples ofparticular values for Z² within this group of preferred embodiments are2-trifluoromethylphenyl, 3-trifluoromethylphenyl,4-trifluoromethylphenyl, 3,5-bis(trifluoromethyl)phenyl, 3-nitrophenyl,3-methoxyphenyl, 3-chlorophenyl, 4-chlorophenyl, 3,4-dichlorophenyl,biphenyl and 2-naphthyl.

In still another group of preferred embodiments, Z² is heteroaryl.Particularly preferred are embodiments in which Z² is pyridyl orpyrimidinyl.

In another group of preferred embodiments, Z¹ and Z² are combined toform a fused 6-membered ring. Particularly preferred are embodiments inZ¹ and Z² are combined to form a fused benzene ring.

Another group of preferred embodiments has the formula (III):

In compounds of formula III, R¹, Y, Z¹ and Z² have the meanings andpreferred groupings provided above.

Yet another group of preferred embodiments is represented by the formula(IV):

In compounds of formula IV, W is (C₁-C₄)alkyl, fluoro(C₁-C₄)alkyl,halogen or nitro, n is an integer from 1-3 and R¹ and Y have themeanings and preferred groupings provided above.

Still another group of preferred embodiments is represented by theformula (V):

wherein R¹, W and n have the meanings and preferred groupings providedabove.

Still another group of preferred embodiments is represented by theformula (VI):

wherein R³, R⁴, Y, Z¹ and Z² have the meanings and preferred groupingsprovided above.

Still another group of preferred embodiments is represented by theformula (VII):

In compounds of formula VII, W is (C₁-C₄)alkyl, fluoro(C₁-C₄)alkyl,halogen or nitro, n is an integer from 1-3 and R³, R⁴ and Y have themeanings and preferred groupings provided above.

Yet another group of preferred embodiments is represented by the formula(VIII):

wherein R³, R⁴, W and n have the meanings and preferred groupingsprovided above. An exemplary compound of formula VIII is:

Preparation of the Compounds

The compounds of the present invention can be prepared using standardsynthetic methods. Scheme 1 illustrates an exemplary method for thepreparation of compounds of structural formula I. One of skill in theart will understand that the synthesis provided below can be modified touse different starting materials and alternate reagents to preparecompounds in the other structural classes. Accordingly, Scheme 1 isexpressed as a non-limiting embodiment.

Analysis of the Compounds

A variety of in vitro and in vivo assays can be used to determinefunctional, chemical, and physical effects of the compounds of thepresent invention. Screening assays may be used to identify compoundsthat (1) modulate the expression and/or activity of UCP3 and (2) can beused as therapeutic agents, e.g., UCP3 upregulators or activators. Thecompounds can be evaluated for modulation of UCP3 expression bymeasuring mRNA levels by, e.g., Northern analysis, QPCR, ribonucleaseprotection analysis, etc. The compounds of the present invention canalso be evaluated for modulation of UCP3 expression by measuring proteinlevels by, e.g., Western analysis, immunoprecipitation and ELISA. Thecompounds can be evaluated for modulation of UCP3 activity by measuringmitochondrial activity, e.g., measuring changes in the mitochondrialmembrane potential.

Table 1 provides QPCR data (ABI 7700 Sequence Detection System) forexemplary compounds of formula I evaluated for UCP3 RNA induction in L6cells from rats. The assay is described in Example 14.4, below.

TABLE 1 UCP3 RNA induction in rat L6 cells. Compound Induction at 10 μM1 ++ 2 ++ 3 ++ 4 ++ 5 ++ 6 ++ 7 ++ 8 — 9 — 10 + 11 + 12 — 13 + 20 + ++denotes greater than 10-fold induction + denotes 10-fold or lessinduction — denotes no induction

UCP3 protein was induced 2- to 3-fold in vivo (skeletal muscle tissuefrom Sprague-Dawley rats) after oral dosing with compound 1 (Westernblot).

Table 2 provides QPCR data (ABI 7700 Sequence Detection System) forexemplary compounds of formula I evaluated for RXR induction in HEK 293cells, following a general protocol, briefly described below. See Naegeret al. (1999) J. Biol. Chem. 274:1875-1878 for a related cell-basedassay.

DNA constructs containing a luciferase reporter plasmid and anotherplasmid which contained an activation domain (e.g., RXRα) weretransiently transfected into HEK 293 cells. The cells were treated withthe compounds and luciferase activity was measured using a luminometer.Fold induction was determined by comparing the luciferase activity oftreated cells to the activity of untreated cells.

TABLE 2 RXRα RNA induction by in HEK 293 cells. Compound at 10 μM at 3μM at 1 μM 1 + + ++ 3 + + + 9 + + + 14 ++ ++ ++ 15 + ++ + 10 + ++ +7 + + + 5 + + + 2 + + + 16 + + + 13 + + + 17 + + + 18 + ++ + 19 + + +8 + + + ++ denotes greater than 5-fold induction + denotes 5-fold orless induction — denotes no inductionCombinatorial Libraries

Combinatorial libraries of compounds of the invention can be screenedfor pharmacological activity in in vitro or in vivo assays.Conventionally, new chemical entities with useful properties aregenerated by identifying a chemical compound (called a “lead compound”)with some desirable property or activity, e.g., UCP3 upregulatingactivity, creating variants of the lead compound, and evaluating theproperty and activity of those variant compounds. However, the currenttrend is to shorten the time scale for all aspects of drug discovery.Because of the ability to test large numbers quickly and efficiently,high throughput screening (HTS) methods are replacing conventional leadcompound identification methods.

In one preferred embodiment, high throughput screening methods involveproviding a library containing a large number of potential therapeuticcompounds (candidate compounds). Such “combinatorial chemical libraries”are then screened in one or more assays to identify those librarymembers (particular chemical species or subclasses) that display adesired characteristic activity. The compounds thus identified can serveas conventional “lead compounds” or can themselves be used as potentialor actual therapeutics.

A combinatorial chemical library is a collection of diverse chemicalcompounds generated by either chemical synthesis or biological synthesisby combining a number of chemical “building blocks” such as reagents.For example, a linear combinatorial chemical library, such as apolypeptide (e.g., mutein) library, is formed by combining a set ofchemical building blocks called amino acids in every possible way for agiven compound length (i.e., the number of amino acids in a polypeptidecompound). Millions of chemical compounds can be synthesized throughsuch combinatorial mixing of chemical building blocks (Gallop et. al.(1994) J. Med. Chem. 37(9):1233-1251).

Preparation and screening of combinatorial chemical libraries is wellknown to those of skill in the art. Such combinatorial chemicallibraries include, but are not limited to, peptide libraries (see, e.g.,U.S. Pat. No. 5,010,175, Furka (1991) Int. J. Pept. Prot. Res.37:487-493, Houghton et. al. (1991) Nature 354: 84-88), peptoidlibraries (PCT Publication No WO 91/19735), encoded peptide libraries(PCT Publication WO 93/20242), random bio-oligomer libraries (PCTPublication WO 92/00091), benzodiazepine libraries (U.S. Pat. No.5,288,514), libraries of diversomers, such as hydantoins,benzodiazepines and dipeptides (Hobbs et. al. (1993) Proc. Nat. Acad.Sci. USA 90:6909-6913), vinylogous polypeptide libraries (Hagihara etal. (1992) J. Amer. Chem. Soc. 114:6568), libraries of nonpeptidylpeptidomimetics with a Beta-D-Glucose scaffolding (Hirschmann et al.(1992) J. Amer. Chem. Soc. 114:9217-9218), analogous organic synthesesof small compound libraries (Chen et. al. (1994) J. Am. Chem. Soc.116:2661), oligocarbamate libraries (Cho et al. (1993) Science 261:1303)and/or peptidyl phosphonate libraries (Campbell et al. (1994) J. Org.Chem. 59:658). See, generally, Gordon et al. (1994) J. Med. Chem.37:1385-1401, nucleic acid libraries (see, e.g., Stratagene Corp.),peptide nucleic acid libraries (see, e.g., U.S. Pat. No. 5,539,083),antibody libraries (see, e.g., Vaughn et. al. (1996) NatureBiotechnology 14(3):309-314), and PCT/US96/10287), carbohydratelibraries (see, e.g., Liang et al. (1996) Science 274:1520-1522, andU.S. Pat. No. 5,593,853), and small organic molecule libraries (see,e.g., benzodiazepines, Baum (1993) C&EN January 18, page 33;isoprenoids, U.S. Pat. No. 5,549,974; pyrrolidines, U.S. Pat. Nos.5,525,735 and 5,519,134; morpholino compounds, U.S. Pat. No. 5,506,337;benzodiazepines, U.S. Pat. No. 5,288,514; and the like).

Devices for the preparation of combinatorial libraries are commerciallyavailable (see, e.g., 357 MPS, 390 MPS, Advanced Chem Tech, LouisvilleKy.; Symphony, Rainin, Woburn Mass.; 433A Applied Biosystems, FosterCity Calif.; 9050 Plus, Millipore, Bedford, Mass.).

A number of well known robotic systems have also been developed forsolution phase chemistries. These systems includes automatedworkstations like the automated synthesis apparatus developed by TakedaChemical Industries, LTD. (Osaka, Japan) and many robotic systemsutilizing robotic arms (Zymate II, Zymark Corporation, Hopkinton Mass.;Orca, Hewlett-Packard, Palo Alto Calif.), which mimic the manualsynthetic operations performed by a chemist. Any of the above devicesare suitable for use with the present invention. The nature andimplementation of modifications to these devices (if any) so that theycan operate as discussed herein will be apparent to persons skilled inthe relevant art. In addition, numerous combinatorial libraries arethemselves commercially available (see e.g., ComGenex, Princeton N.J.;Asinex, Moscow, Russia; Tripos, Inc., St. Louis Mo.; ChemStar, Ltd,Moscow, Russia; 3D Pharmaceuticals, Exton Pa.; Martek Biosciences,Columbia Md.; etc.).

High Throughput Screening

High throughput assays for the presence, absence, quantification, orother properties of particular compounds may be used to test acombinatorial library that contains a large number of potentialtherapeutic compounds (potential modulator compounds). The assays aretypically designed to screen large chemical libraries by automating theassay steps and providing compounds from any convenient source toassays, which are typically run in parallel (e.g., in microtiter formatson microtiter plates in robotic assays). Preferred assays detectenhancement or upregulation of UCP3 gene transcription.

High throughput screening systems are commercially available (see e.g.,Zymark Corp., Hopkinton Mass.; Air Technical Industries, Mentor Ohio;Beckman Instruments, Inc., Fullerton Calif.; Precision Systems, Inc.,Natick Mass.; etc.). These systems typically automate entire procedures,including all sample and reagent pipetting, liquid dispensing, timedincubations, and final readings of the microplate in detector(s)appropriate for the assay. These configurable systems provide highthroughput and rapid start up as well as a high degree of flexibilityand customization. The manufacturers of such systems provide detailedprotocols for various high throughput systems. Thus, for example, ZymarkCorp. provides technical bulletins describing screening systems fordetecting the modulation of gene transcription, ligand binding, and thelike.

The following examples are offered by way of illustration and not by wayof limitation.

EXAMPLES

Reagents and solvents used below can be obtained from commercial sourcessuch as Aldrich Chemical Co. (Milwaukee Wis., USA), Fisher Scientific(Loughborough UK), Acros (Loughborough UK) and ROMIL (Cambridge UK).

Method A Preparation of Thiazol-2-yl-5-Methylpyrazol-4-Carboxylic AcidsPreparation of Hydroxymethylene Esters

Crude ethyl 2-(ethoxymethylene)-3-oxobutanoate (244 g) was stirred whileadding 1.5 L of saturated aqueous copper (II) acetate over one hour.Stirring was continued for three h and the blue solid was collected,washed well with water then diethyl ether and dried to constant weightat room temperature to yield 197 g.

The solid was added to 1.4 L of ether and stirred while adding dropwiseover two h 750 mL 1M sulphuric acid. The ether layer was dried (sodiumsulphate), evaporated and distilled from a water bath at 75° C. using anoil pump giving product as colorless oil, b.p. 38° C./0.3 mb. Yield 135g pure material shown by NMR to be a 9:1 mixture of geometric isomers.

Preparation of 5-Methylpyrazol-1-ylthioamides

Thiosemicarbazide (18.2 g) was stirred with 300 mL ethanol undernitrogen below −10° C. while adding dropwise 31.6 g ester (1). Themixture was stirred overnight at room temperature giving a colorlesssuspension.

The solid was collected, washed with ethanol and dried in air, then at20 mb at room temperature, to a constant weight of 40.9 g. Material wasstored at 4° C.

Preparation of Alkyl Thiazol-2-yl-5-methylpyrazol-4-carboxylates

3-(Trifluoromethyl)acetophenone (7.73 g) in 50 mL dry diethyl ether wasstirred while adding rapidly 2.32 mL bromine. The ether boiled gentlyand was stirred for 2 h after the reaction had subsided. Stirring with50 mL water and adding sodium hydrogen carbonate in small portions untilno further effervescence gave a colorless solution of ethyl1-[4-(3-(trifluoromethyl)phenyl)thiazol-2-yl]-5-methylpyrazole-4-carboxylate(11), which was washed with brine, dried (sodium sulphate) and useddirectly.

Preparation of Thiazol-2-yl-5-Methylpyrazol-4-carboxylic Acids

Ester 11 (8.72 g) in 200 mL ethanol was treated with a solution of 2.2 gpotassium hydroxide in 6 mL water and heated briefly when all went intosolution. The potassium salt of the product separated rapidly. Water(200 mL) was added, the ethanol removed by distillation and the solutionallowed to cool. Acidification with 3.9 mL concentrated hydrochloricacid and boiling briefly gave solid which was collected, washed withwater and dried well in air.

The solid was dissolved in 200 mL toluene/50 nL acetone by heating andthe acetone was then removed by distillation. On cooling the solid wascollected, washed with toluene and dried in air, then at 110° C./20 mb,to give a constant weight of 7.56 g of1-[4-(3-(trifluoromethyl)phenyl)thiazol-2-yl]-5-methylpyrazole-4-carboxylicacid (1).

Method B Preparation of Thiazol-2-ylpyrazole-4-carboxylic acidsPreparation of Pyrazol-1-ylthioamides

Ethyl 3,3-diethoxypropionate (Acros, 49.7 g) in 100 mL dry diethyl etherwas added to a stirred suspension of 13.5 g 60% sodium hydridedispersion in oil in 300 mL ether/200 mL ethyl formate (both previouslydried over 4 Å molecular sieves overnight) dropwise with ice/watercooling over 2 h. After 7 h the mixture was stirred at room temperatureovernight. Hydrogen was still being evolved so the mixture was stirredfor a further 48 h. Addition to water (200 mL) and washing with ether toremove starting ester gave a solution which was carefully brought to pH3 using 3M-hydrochloric acid. Extraction with dichloromethane (3×200mL), drying (sodium sulphate) and evaporating gave crudeethoxycarbonylmalondialdehyde as a pale orange oil which was useddirectly.

The above oil (15 g) in 50 mL ethanol was added at −10° C. to asuspension of 9.1 g finely powdered thiosemicarbazide in 150 mL ethanol.After stirring at room temperature for three days the solid wascollected, washed with ethanol and dried in air giving an orange powder.The NMR spectrum was complex as usual for thioamides, but HPLC-MS gave asingle peak having the correct mass.

Preparation of Alkyl Thiazol-2-ylpyrazole-4-carboxylates

Ethyl 3,3-diethoxypropionate (Acros, 49.7 g) in 100 mL dry diethyl etherwas added to a stirred suspension of 13.5 g 60% sodium hydridedispersion in oil in 300 mL ether/200 mL ethyl formate (both previouslydried over 4 Å molecular sieves overnight) dropwise with ice/watercooling over 2 h. After 7 h the mixture was stirred at room temperatureovernight. Hydrogen was still being evolved so the mixture was stirredfor a further 48 h. Addition to water (200 mL) and washing with ether toremove starting ester gave a solution which was carefully brought to pH3 using 3M-hydrochloric acid. Extraction with dichloromethane (3×200mL), drying (sodium sulphate) and evaporating gave crudeethoxycarbonylmalondialdehyde as a pale orange oil which was useddirectly.

The above oil (15 g) in 50 mL ethanol was added at −10° C. to asuspension of 9.1 g finely powdered thiosemicarbazide in 150 mL ethanol.After stirring at room temperature for three days the solid wascollected, washed with ethanol and dried in air giving an orange powder.The NMR spectrum was complex as usual for thioamides but HPLC-MS gave asingle peak having the correct mass.

3-Trifluoromethylphenacyl bromide was prepared as in Method A from 5.15g of the acetophenone and the ether solution added to a suspension of5.45 g of thioamide prepared as above in 200 mL ethanol/5 mL pyridineand heated under reflux in a nitrogen atmosphere overnight.

The deep brown mixture was filtered and the solid discarded. Thefiltrate was evaporated and the residue treated with ethanol giving 1.1g deep yellow-brown solid with the correct NMR spectrum. Dissolving in50 mL dichloromethane, treating with charcoal, filtering, adding 30 mLethanol and evaporating the dichloromethane gave, on cooling, palered-brown leaflets (0.77 g) of ethyl1-[4-(3-(trifluoromethyl)phenyl)thiazol-2-yl]pyrazole-4-carboxylate.m.p. 158.7-159.1° C.

Preparation of Thiazol-2-ylpyrazole-4-carboxylic acids

The above ester (0.55 g) in 30 mL ethanol was treated with a solution of0.5 g potassium hydroxide in 2 mL water and heated under reflux for twoh. The ethanol was evaporated, the residue dissolved in 30 mL water,filtered and the pale yellow filtrate acidified with hydrochloric acid.The white solid was collected, washed well with water, dried briefly inair and taken up in 20 mL acetone. Addition of 40 mL toluene and heatingto remove acetone and water resulted on cooling in nearly colorlesssolid. This was collected, washed with toluene and dried in air to aconstant weight of 0.38 g of1-[4-(3-(trifluoromethyl)phenyl)thiazol-2-yl]pyrazole-4-carboxylic acid(6).

Method C Preparation of Thiazol-2-ylpyrazole-4-carboxylic acidsPreparation of 5-(Trifluoromethyl)pyrazol-1-ylthioamides

Ethyl 2-(ethoxymethylene)-3-oxo-4,4,4-trifluorobutyrate (10.5 g) wasadded below −10° C. to a stirred suspension of thiosemicarbazide (3.98g) in 60 mL ethanol and stirred overnight.

The resulting solid was collected, washed twice with ethanol and driedat 30° C./20 mb to constant weight. The product was stored at 4° C.

Preparation of AlkylThiazol-2-yl-5-(trifluoromethyl)pyrazole-4-carboxylates

3,5-Bis(trifluoromethyl)acetophenone (2.42 g) was converted into thephenacyl bromide in 15 mL ether using 0.50 mL bromine as in Method A.The purified ether solution was added to a stirred suspension ofthioamide prepared as above (2.5 g) in 50 mL ethanol and 1.0 mL pyridineand heated under reflux after allowing the ether to escape for 3 h.

The yellow-brown solution was evaporated under vacuum, the residuetreated with water then extracted into ether. Pyridine was removed bywashing with saturated copper (II) acetate solution twice then with 0.5Mhydrochloric acid twice. Drying (sodium sulphate) and evaporating gave4.3 g deep brown semi-solid which was dissolved in 100 mL hotcyclohexane and treated with charcoal.

Flash chromatography (silica) using cyclohexane removed non-polarimpurities and product-containing fractions were eluted with 20%ether/80% 40-60 petrol. Evaporation gave 1.65 g brown crystalline solidcontaining 90% product as shown by NMR. Trituration of the solid withethanol gave pale yellow solid which was washed and dried in air to aconstant weight of 1.0 g. m.p. 126.0-126.4° C.

Preparation of Thiazol-2-yl-5-(trifluoromethyl)pyrazole-4-carboxylicAcids

Ethyl1-[4-(3,5-(bistrifluoromethyl)phenyl)thiazol-2-yl]-5-trifluoromethyl)pyrazole-4-carboxylate(0.8 g) was hydrolysed using 0.3 g potassium hydroxide/1 mL water in 15mL ethanol as described in Method B, giving 0.37 g of1-[4-(3,5-(bistrifluoromethyl)phenyl)thiazol-2-yl]-5-trifluoromethyl)pyrazole-4-carboxylicacid (13) as a cream crystalline solid.

Example 1

1-[4-(3-(Trifluoromethyl)phenyl)thiazol-2-yl]-5-methylpyrazole-4-carboxylicacid (1) was prepared according to Method A. m.p. 226.7-227.5° C.

Example 2

1-[4-(4-(Trifluoromethyl)phenyl)thiazol-2-yl]-5-methylpyrazole-4-carboxylicacid (2) was prepared according to Method A. m.p. 273.0-274.0° C.

Example 3

1-[4-(3,4-Dichlorophenyl)thiazol-2-yl]-5-methylpyrazole-4-carboxylicacid (3) was prepared according to Method A. m.p. 278.0-278.6° C.

Example 4

1-[4-(3-Chlorophenyl)thiazol-2-yl]-5-methylpyrazole-4-carboxylic acid(4) was prepared according to Method A. m.p. 258.0-258.3° C.

Example 5

1-[4-(3-(Trifluoromethyl)phenyl)thiazol-2-yl]-5-trifluoromethylpyrazole-4-carboxylicacid (5) was used as purchased.

Example 6

1-[4-(3-(Trifluoromethyl)phenyl)thiazol-2-yl]pyrazole-4-carboxylic acid(6) was prepared according to Method B. m.p. 229.1-229.6° C.

Example 7

1-[4-(2-(Trifluoromethyl)phenyl)thiazol-2-yl]-5-methylpyrazole-4-carboxylicacid (7) was prepared according to Method A. m.p. 215.5-216.5° C.

Example 8

Ethyl 1-[4-(3-nitrophenyl)thiazol-2-yl]-5-methylpyrazole-4-carboxylate(8) was prepared according to Method A.

Example 9

1-[4-(2-Naphthyl)thiazol-2-yl]-5-methylpyrazole-4-carboxylic acid (9)was prepared according to Method A. m.p. 273.1-274.3° C.

Example 10

1-[4-(3,5-Bis(trifluoromethyl)phenyl)thiazol-2-yl]-5-methylpyrazole-4-carboxylicacid (10) was prepared according to Method A. m.p. 260.9-261.4° C.

Example 11

Ethyl1-[4-(3-(trifluoromethyl)phenyl)thiazol-2-yl]-5-methylpyrazole-4-carboxylate(11) was prepared according to Method A. m.p. 138-138.5° C.

Example 12

1-[4-(3-Nitrophenyl)thiazol-2-yl]-5-trifluoromethylpyrazole-4-carboxylicacid (12) was used as purchased.

Example 13

1-[4-(3,5-Bis(trifluoromethyl)phenyl)thiazol-2-yl]-5-(trifluoromethyl)pyrazole-4-carboxylicacid (13) was prepared according to Method C. m.p. 204.0-204.7° C.

Example 14 In Vitro Evaluation of Compounds of the Invention Example14.1 Mitochondrial Protein Preparation from Skeletal Muscle

1. Isolate rat or mouse skeletal muscle and snap freeze in liquidnitrogen. Grind into a fine powder using mortar and pestle and transferto an eppendorf tube.

2. Resuspend skeletal muscle in Buffer 1 (0.15 mM MgCl₂, 10 mM KCl, 10mM Tris-HCl, pH 6.7, stored at 4° C.).

3. Place resuspended cells on ice for 2 min. Homogenize using eitherPotter-Eleuhjem Teflon homogenizer and glass dounce or using hand heldhomogenizer for 2 min.

4. Add sucrose (1 M) to a final concentration of 0.25 M and spin at 1500g (4000 rpm in microfuge) for 3 min. to remove nuclei.

5. Take the supernatant fraction and pellet the mitochondria bycentrifugation at 10000 rpm for 10 min.

6. Resuspend pellet in 500 μl Buffer 2 (0.15 mM MgCl₂, 0.25 mM Sucrose,10 mM Tris-HCl, pH 6.7, stored at 2-4° C.).

7. Spin at 10,000 rpm for 10 min.

8. Repeat steps 6 and 7.

9. Resuspend final mitochondrial pellet in 2 7 μl Buffer 3 (0.25 MSucrose, 10 mM Tris-acetate, pH 7.0, 3 μl 10× protease inhibitorcocktail (BMB), stored at 4° C.).

10. Determine protein concentration with BioRad protein assay dye(Bradford Reagent). 1 μl protein in 100 μl 1× Bradford Reagent. OD595with BSA standard curve (1 μg/μl-50 μg/μl).

11. Store at −80° C.

For antiUCP3 Westerns use 60 μg protein. Run precast 10% gels until dyefront is about 1 cm from bottom of gel. Western with anti-UCP3 antibodyfrom Alpha Diagnostics.

Example 14.2 mUCP3 QPCR Protocol

1. Plate 125,000 C2C12 cells per well (24 well plate) in 20% FBS/DMEM.Incubate for 2 days.

2. Add compounds in 20% FBS/DMEM to final concentration of 10 μM.Incubate for 18 h. 3 wells per compound.

3. Prepare RNA from cells:

Aspirate media and lyse cells directly on plate by adding 500 μL ofTrizol Reagent to each well and passing the lysate several times througha pipette. Transfer lysate to eppendorf tubes (combining the lysate fromidentical compound treatments). Add 200 mL of chloroform to the 1 mL oflysate. Vortex to mix and incubate at room temp. for 2-3 min. Spinsamples in a microfuge at 12,000 rpm for 15 min. at 4° C. Transfer theaqueous phase to a fresh tube. Precipitate the RNA from the aqueousphase by adding 500 μL of isopropanol and mixing well. Incubate at −20°C. for at least 15 min. Pellet the RNA by spinning at 12,000 rpm for 10min. at 4° C. Remove the supernate and wash the RNA pellet with 1 mL of75% ethanol. Spin at 12,000 rpm for 10 min. at 4° C. Remove thesupernate and allow pellet to air dry briefly. Dissolve RNA in 15 μL ofRNase-free water. Store at −80° C.

4. Reverse Transcription Reaction Mixture (50 μL reactions in 96 wellPCR plates): TaqMan Reverse Transcription Reagents (PE catalog#N808-0234):

Component Volume/Tube (μL) Final Concentration RNase-free water 15 1 10XTaqMan RT Buffer 5 1X 25 mM MgCl₂ 11 5 Mm deoxyNTPs mixture 10 500 μM ofeach Random Hexamer 2.5 2.5 μM RNase Inhibitor 1.0 0.4 U/μL MultiScribeRT 1.25 1.25 U/μL RNA 5 50 μLRT Cycling Parameters

-   25° C. 10 min-   48° C. 30 min-   95° C. 5 min

5. QPCR (50 μL reactions in 96 well MicroAmp Optical Plate): TaqMan PCRCore Reagents Kit (PE catalog #N808-0228): OD cDNA and dilute to 5ng/μL. All samples run in duplicate with mUCP3 and m18S or GAPDHprobe/primers.

Component Volume/Tube (μL) Final Concentration RNase-free water 19.3 —10X TaqMan Buffer A 5 1X 25 mM MgCl₂ 10 5 mM 10 mM dATP 1 200 μM 10 mMdCTP 1 200 μM 10 mM dGTP 1 200 μM 20 mM dUTP 1 400 μM 10 μM ForwardPrimer 0.3/0.15 60 nM/30 nM 10 μM Reverse Primer 0.3/0.15 60 nM/30 nM 10uM Probe 0.3/0.15 60 nM/30 nM AmpErase UNG 0.5 0.01 U/μL AmpliTaq GoldDNA Poly. 0.25 0.025 U/μLAdd 40 μL of Reaction Mix to 10 μL of diluted cDNA (50 ng total) on QPCRplatePCR Cycling Parameters

50° C. 2 min 95° C. 10 min 40 cycles: 95° C. 15 sec 58° C. 1.5 minQPCR Primers/Probes

mUCP3: Forward Primer: 5′ TGACCTGCGCCCAGC 3′ (SEQ ID NO:1) ReversePrimer: 5′ CCCAGGCGTATCATGGCT 3′ (SEQ ID NO:2) Probe: 5′CACGGATGTGGTGAAGGTCCGATTT 3′ (SEQ ID NO:3) GAPDH: Forward Primer: 5′AAAGTGGAGATTGTTGCCAT 3′ (SEQ ID NO:4) Reverse Primer: 5′TTGACTGTGCCGTTGAATT 3′ (SEQ ID NO:5) Probe: 5′CATGTTCCAGTATGACTCCACTCACG 3′ (SEQ ID NO:6) m18S: Forward Primer: 5′CCCTGCCCTTTGTACACACC 3′ (SEQ ID NO:7) Reverse Primer: 5′CGATCCGAGGGCCTCACTA 3′ (SEQ ID NO:8) Probe: 5′CCCGTCGCTACTACCGATTGGATGGT 3′ (SEQ ID NO:9)

Example 14.3 Rat UCP3 (Skeletal Muscle) QPCR Protocol

1. Harvest skeletal muscle from rat, snap freeze in liquid nitrogen.

2. Grind skeletal muscle tissue in mortar and pestle until it is a finepowder.

3. Prepare RNA:

-   -   (1) Add 5 mL of Trizol Reagent (Gibco BRL) and homogenize using        Virtis Cyclone generator for 50 seconds.    -   (2) Add another 5 mL Trizol reagent after homogenization.    -   (3) Add 2 mL of chloroform to the 10 mL homogenate. Vortex to        mix and incubate at room temp. for 2-3 min. Spin samples in a        microfuge at 12,000 rpm for 15 min. at 4° C.    -   (4) Transfer the aqueous phase to a fresh tube. Precipitate the        RNA from the aqueous phase by adding 5 mL of isopropanol and        mixing well. Incubate at −20° C. for at least 15 min. Pellet the        RNA by spinning at 12,000 pm for 10 min. at 4° C.    -   (5) Remove the supernatant and wash the RNA pellet with 5 mL of        75% ethanol. Spin at 10,000 rpm for 5 min. at 4° C.    -   (6) Remove the supernatant and allow pellet to air dry briefly.        Dissolve RNA in 100 μL of RNase-free water. Store at −80° C.        Electrophorese some RNA to check quality.

4. QPCR (50 μL reactions in 96 well MicroAmp Optical Plate):

TaqMan Gold RT-PCR Reagents (PE catalog #N808-0232): OD RNA and diluteto 5 ng/μL in RNase-free water. All samples run in duplicate with UCP3and m18S probe/primers.

Component Volume/Tube (μL) Final Concentration RNase-free water 18 — 10XTaqMan Buffer A 5 1X 25 mM MgCl₂ 10 5 mM 10 mM dATP 1 200 μM 10 mM dCTP1 200 μM 10 mM dGTP 1 200 μM 20 mM dUTP 1 400 μM 10 uM Forward Primer0.3/0.15 60 nM/30 nM 10 uM Reverse Primer 0.3/0.15 60 nM/30 nM 10 uMProbe 0.3/0.15 60 nM/30 nM MultiScribe RT 0.25 0.25 U/μL AmpliTaq GoldDNA Poly. 0.25 0.025 U/μL RNase Inhibitor 1 0.4 U/μLAdd 40 μL of Reaction Mix to 10 μL of diluted RNA (50 ng total) on QPCRplatePCR Cycling Parameters

-   48° C. 30 min.-   95° C. 10 min.-   40 cycles: 95° C. 15 sec.-   58° C. 1.5 min.    QPCR Primers/Probes

rUPC3: Forward Primer: 5′ GGCAGTGACCTGTGCTCAAC 3′(rU3-F) (SEQ ID NO:10)Reverse Primer: 5′ CCCAGGCGTATCATGGCT 3′(mU3-R) (SEQ ID NO:11) Probe: 5′6FAM-CACGGATGTGGTGAAGGTCCGATTT- (SEQ ID NO:12) TAMRA 3′ (mU3-Probe)m18S: Forward Primer: 5′ CCCTGCCCTTTGTACACACC 3′ (SEQ ID NO:13) ReversePrimer: 5′ CGATCCGAGGGCCTCACTA 3′ (SEQ ID NO:14) Probe: 5′CCCGTCGCTACTACCGATTGGATGGT 3′ (SEQ ID NO:15)

Example 14.4 Rat UCP3 (L6 cells) QPCR Protocol

1. (Day1) Plate 300,000 L6 cells per well (24 well plate) in MinimumEssential Medium-Alpha Medium with 2% FBS and Antibiotic/AntimycoticSolution added. Incubate for 2 days and change to fresh medium (Day3).Incubate 1 more day.

2. (Day 4) Add compound at desired concentration. Incubate for 18-20 h.2 wells per compound. 4-wells of DMSO controls per plate.

3. (Day 5) Prepare RNA from cells:

-   -   (1) Aspirate media and lyse cells directly on plate by adding        500 μL of Trizol Reagent (Gibco BRL) to each well and passing        the lysate several times through a pipette. Transfer lysate to        eppendorf tubes (combining the lysate from identical compound        treatments).    -   (2) Add 200 μL of chloroform to the 1 mL of lysate. Vortex to        mix and incubate at room temp. for 2-3 min. Spin samples in a        microfuge at 12,000 rpm for 15 min. at 4° C.    -   (3) Transfer the aqueous phase to a fresh tube. Precipitate the        RNA from the aqueous phase by adding 500 μL of isopropanol and        mixing well. Incubate at −20° C. for at least 15 min. Pellet the        RNA by spinning at 12,000 rpm for 10 min. at 4° C.    -   (4) Remove the supernatant and wash the RNA pellet with 500 μL        of 75% ethanol. Spin at 10,000 rpm for 5 min. at 4° C.    -   (5) Remove the supernatant and allow pellet to air dry briefly.        Dissolve RNA in 15 μL of RNase-free water. Store at −80° C.        Electrophorese some RNA to check quality.

4. QPCR (50 μL reactions in 96 well MicroAmp Optical Plate): TaqMan GoldRT-PCR Reagents (PE catalog #N808-0232): OD RNA and dilute to 5 ng/μL inRNase-free water. All samples run in duplicate with UCP3 and m18Sprobe/primers

Component Volume/Tube (μL) Concentration RNase-free water 18 — 10XTaqMan Buffer 5 1X 25 mM MgCl₂ 10 5 mM 10 mM dATP 1 200 μM 10 mM dCTP 1200 μM 10 mM dGTP 1 200 μM 20 mM dUTP 1 400 μM 10 uM Forward Primer0.3/0.15 60 nM/30 nM 10 uM Reverse Primer 0.3/0.15 60 nM/30 nM 10 uMProbe 0.3/0.15 60 nM/30 nM MtiScribe RT 0.25 0.25 U/μL AmpliTaq Gold DNAPoly. 0.25 0.025 U/μL RNase Inhibitor 1 0.4 U/μLAdd 40 μL of Reaction Mix to 10 μL of diluted RNA (50 ng total) on QPCRplatePCR Cycling Parameters

-   48° C. 30 min.-   95° C. 10 min.-   40 cycles: 95° C. 15 sec.-   58° C. 1.5 min.    QPCR Primers/Probes

rUPC3: Forward Primer: 5′ GGCAGTGACCTGTGCTCAAC 3′(rU3-F) (SEQ ID NO:16)Reverse Primer: 5′ CCCAGGCGTATCATGGCT 3′(mU3-R) (SEQ ID NO:17) Probe: 5′6FAM-CACGGATGTGGTGAAGGTCCGATTT- (SEQ ID NO:18) TAMRA 3′ (mU3-Probe)m18S: Forward Primer: 5′ CCCTGCCCTTTGTACACACC 3′ (SEQ ID NO:19) ReversePrimer: 5′ CGATCCGAGGGCCTCACTA 3′ (SEQ ID NO:20) Probe: 5′CCCGTCGCTACTACCGATTGGATGGT 3′ (SEQ ID NO: 21)

All publications and patent applications cited in this specification areherein incorporated by reference as if each individual publication orpatent application were specifically and individually indicated to beincorporated by reference. Although the foregoing invention has beendescribed in some detail by way of illustration and example for purposesof clarity of understanding, it will be readily apparent to those ofordinary skill in the art in light of the teachings of this inventionthat certain changes and modifications may be made thereto withoutdeparting from the spirit or scope of the appended claims.

1. A method for treating obesity or diabetes, comprising administeringto a subject in need thereof a therapeutically effective amount of acompound of formula (I):

or a pharmaceutically acceptable salt or prodrug thereof, wherein X isselected from the group consisting of CO₂R¹ and C(O)NR¹R²; Y is selectedfrom the group consisting of hydrogen, (C₁-C₄)alkyl, fluoro(C₁-C₄)alkyl,aryl, heteroaryl, halogen, NR³R⁴ and CO₂R³; Z¹ is selected from thegroup consisting of hydrogen, (C₁-C₄)alkyl and halogen; Z² is selectedfrom the group consisting of aryl and heteroaryl, or Z¹ and Z² may becombined to form a fused 6-membered ring; and R¹, R², R³ and R⁴ areindependently selected from the group consisting of hydrogen,(C₁-C₄)alkyl, carboxy(C₁-C₄) alkyl and aryl.
 2. The method of claim 1,wherein said compound has the formula (II):

or a pharmaceutically acceptable salt or prodrug thereof, wherein X isselected from the group consisting of CO₂R¹ and C(O)NR¹R²; Y is selectedfrom the group consisting of hydrogen, (C₁-C₄alkyl, fluoro(C₁-C₄)alkyl,aryl, heteroaryl, halogen, NR³R⁴ and COR₂R³; Z¹ is selected from thegroup consisting of hydrogen, (C₁-C₄alkyl and halogen; Z² is selectedfrom the group consisting of aryl and heteroaryl, or Z¹ and Z² may becombined to form a fused 6-membered ring; and R¹, R², R³ and R⁴ areindependently selected from the group consisting of hydrogen,(C₁-C₄)alkyl, carboxy(C₁-C₄)alkyl and aryl.
 3. The method of claim 1,wherein X is CO₂H.
 4. The method of claim 1, wherein Y is hydrogen.(C₁-C₄) alkyl or fluoro (C₁-C₄)alkyl.
 5. The method of claim 1, whereinZ¹ is hydrogen.
 6. The method of claim 1, wherein Z² is phenyl,naphthyl, pyridyl or pyrimidinyl.
 7. The method of claim 1, wherein Z²is phenyl and X is CO₂H.
 8. The method of claim 1, wherein Z² is phenyland Y is (C₁-C₄alkyl or fluoro(C₁-C₄)alkyl.
 9. The method of claim 1,wherein Z¹ and Z² are combined to form a fused benzene ring.
 10. Themethod of claim 2, wherein said compound has the formula (III):

or a pharmaceutically acceptable salt or prodrug thereof, wherein Y isselected from the group consisting of hydrogen, (C₁-C₄)alkyl,fluoro(C₁-C₄)alkyl, aryl, heteroaryl, halogen, NR³R⁴ and CO₂R³; Z¹ isselected from the group consisting of hydrogen, (C₁-C₄)alkyl andhalogen; Z² is selected from the group consisting of aryl andheteroaryl, or Z¹ and Z² may be combined to form a fused 6-memberedring; and R¹, R³ and R⁴ are independently selected from the groupconsisting of hydrogen, (C₁-C₄)alkyl, carboxy(C₁-C₄)alkyl and aryl. 11.The method of claim 10, wherein R¹ is hydrogen.
 12. The method of claim10, wherein Z² is phenyl.
 13. The method of claim 12, wherein saidcompound has the formula (IV):

or a pharmaceutically acceptable salt or prodrug thereot wherein Y isselected from the group consisting of hydrogen, (C₁-C₄)alkyl,fluoro(C₁-C₄alkyl, aryl, heteroaryl, halogen, NR³R⁴ and CO₂R³; R¹, R²and R⁴ are independently selected from the group consisting of hydrogen,(C₁-C₄)alkyl, carboxy(C₁-C₄)alkyl and aryl; W is (C₁-C₄)alkyl,fluoro(C₁-C₄)alkyl, halogen or nitro; and n is an integer from 1 to 3.14. The method of claim 13, wherein Y is (C₁-C₄)alkyl.
 15. The method ofclaim 2, wherein said compound has the formula (VI):

or a pharmaceutically acceptable salt or prodrug thereof, wherein Y isselected from the group consisting of hydrogen, (C₁-C₄)alkyl,fluoro(C₁-C₄)alkyl, aryl, heteroaryl, halogen, NR³R⁴ and CO₂R³; Z¹ isselected from the group consisting of hydrogen, (C₁-C₄)alkyl andhalogen; Z² is selected from the group consisting of atyl andheteroaryl, or Z¹ and Z² may be combined to form a fused 6-memberedring; and R¹, R², R³ and R⁴ are independently selected from the groupconsisting of hydrogen, (C₁-C₄)alkyl, carboxy(C₁-C₄)alkyl and aryl. 16.The method of claim 15, wherein Z² is phenyl.
 17. The method of claim16, wherein said compound has the formula (VII):

or a pharmaceutically acceptable salt or prodrug thereof, wherein Y isselected from the group consisting of hydrogen, (C₁-C₄)alkyl,fluoro(C₁-C₄)alkyl, aryl, heteroaryl, halogen, NR³R⁴ and CO₂R³; R¹, R²,R³ and R⁴ are independently selected from the group consisting ofhydrogen, (C₁-C₄)alkyl, carboxy(C₁-C₄)alkyl and aryl; W is (C₁-C₄)alkyl,fluoro(C₁-C₄)alkyl, halogen or nitro; and n is an integer from 1 to 3.18. The method of claim 17, wherein Y is (C₁-C₄)alkyl.
 19. The method ofclaim 1, wherein said compound is


20. The method of claim 1, wherein said compound is administered incombination with a therapeutic agent selected from the group consistingof a β₃ adrenergic receptor agonist, a leptin, a lepuin derivative, aneuropeptide Y antagonist insulin and derivatives thereof, ahypoglycemic agent, an antihyperglyeemic agent, an czglucosidaseinhibitor, an insulin sensitizer, an RXR agonist, a cholesterol loweringagent, a calcium channel blocker, interferon alpha, interferon beta, aDNA-alkylating agent, an antirnetabolite, a microtubule disruptor, a DNAintercalator, a DNA synthesis inhibitor and a hormone.
 21. The method ofclaim 1, wherein said administering is oral or parenteral.